Taste transduction begins when sapid stimuli interact with the apical membrane of receptor cells, causing an increase in intracellular Ca 2+. This ultimately results in release of transmitter and activation of gustatory afferent fibers. Several G protein-coupled taste receptors have been identified: T2Rs respond to bitter, T1R2/T1R3 to sweet, and T1R1/T1R3 and taste-mGluR4 to umami stimuli. Further, these receptors all activate PLCbeta2, causing Ca2+ release from intracellular stores and Ca2+ influx via the cation channel TrpM5, although the precise role of each of these signaling components is not understood. Surprisingly, PLCbeta2 is present primarily in Type II taste cells, which lack conventional synapses with afferent nerve fibers. Thus, how these taste qualities are transmitted to the nervous system is unclear. Our goal for the proposed experiments is to elucidate the link between the initial events of taste transduction and activation of afferent nerve fibers. Experiments are proposed to determine the relative role of T1R1/T1R3 and taste-mGluR4 in umami signaling, their downstream signaling effectors, and how responses to umami and bitter stimuli are communicated to the nervous system. Aim 1 uses immunocytochemistry and in situ hybridization to determine whether the two umami receptors are co-localized, and Ca 2+ imaging and patch clamp recording to determine the response profile of each receptor, whether the response is potentiated by 5'-ribonucleotides, and the conductance(s) that are affected. Aim 2 utilizes Ca2+ imaging and electrophysiology to determine the downstream signaling effectors important for umami and bitter taste transduction, including the roles of PLCbeta2, TrpM5, and alpha-gustducin. In aim 3, we will test whether Type III taste cells are required for communicating bitter, umami, or sweet taste information to the nervous system with the production of two new transgenic mice. Both will involve targeting of genes to the BDNF locus, since BDNF is selectively expressed in adult taste cells with synapses: (1) BDNF-GFP mice, which will express GFP in all taste cells with synapses, so they can be identified for physiological recording, and (2) BDNF-DT mice, which will undergo selective ablation of synaptically-connected taste cells, so their role in taste information processing can be directly determined.